There is not an experimental model of localized cutaneous leishmaniasis (LCL) caused by Leishmania (Leishmania) mexicana. The aim of the present study was to characterize the clinical and histological features of Peromyscus yucatanicus experimentally infected with L. (L.) mexicana. A total of 54 P. yucatanicus (groups of 18) were inoculated with 1x106 promastigotes of L. (L.) mexicana in the base of the tail. They were euthanized at three and six months post experimental infection. The control group was inoculated with RPMI-1640. The predominant clinical sign observed was a single ulcerated lesion in 27.77% (5/18) and in 11.11% (2/18) P. yucatanicus at three and six months respectively. The histological pattern described as chronic granulomatous inflammation with or without necrosis was found in 7/7 (100%) biopsies of euthanized P. yucatanicus at three (n = 5) and six (n = 2) months, respectively. These results resembled clinical and histological features caused by L. (L.) mexicana in humans, and support the possibility to employ P. yucatanicus as a novel experimental model to study LCL caused by this parasite.

Localized cutaneous leishmaniasis (LCL) known as "chiclero´s ulcer" (collector of natural chewing gum) in southern Mexico, was described by Seidelin in 191231. Since then, the forest region of the Yucatan peninsula has been recognized as an endemic focus of LCL3,6,32. Leishmania (Leishmania) mexicana Biagi, 1953, emend. Garnham, 1962, is the main agent causing CL in this focus; however, L. (Viannia) braziliensis has also been isolated in a few cases9,10.

The clinical features of "chiclero´s ulcer" have been studied and it is characterized predominantly by cartilage involvement of the pinna of the ear7,16,39. The most frequently observed presentation is a single, ulcerated, rounded small lesion, with elevated borders and necrotic center, located on the ear, with an evolution time of less than three months, and with neither cutaneous metastases nor lymphatic nor mucosal involvement4. Criteria for scars resulting from healed lesions of humans with CL caused by Leishmania (Viannia) braziliensis include: no history of trauma, round or oval shape, smooth surface, depressed, and pigment change, hypopigmentation38. No differences have been observed in scars from LCL caused by L. (L.) mexicana (unpublished data).

The histopathology of LCL due to Leishmania (Leishmania) mexicana in the Yucatan Peninsula, Mexico, has also been described. Type III and IV of Magalhaes' classification21 have been found as the most common patterns in 21/73 (28.7%) and 32/73 (43.8%) respectively of our LCL cases caused by L. (L.) mexicana5.

The cutaneous leishmaniasis laboratory mouse model using C57BL/6 (resistant) and BALB/c (susceptible) infected with L. major is the most employed system for analyzing the regulation of the immune response against this pathogen only contained by a cell-mediated response19,30. However, C57BL/6 mice inoculated with L. (L.) mexicana developed a chronic disease2. Although the L. major mouse model is considered by many to be the best small animal immunological model for CL, concern about its relevance suggests other models should be developed. In particular, because laboratory mice are usually reduced to homozygosity, are hybrids of three species (Mus musculus musculus, M. m. domesticus, M. m. castaneus)37 and are not natural reservoirs of leishmaniasis parasites, the use of other, wild-type species that are naturally infected should be considered as alternative models.

In Campeche, three rodents have been identified as primary reservoirs of L. (L.) mexicana: Ototylomys phyllotis Merriam, 1901; Peromyscus yucatanicus J.A. Allen and Chapman, 1897; and Heteromys gaumeri J.A. Allen and Chapman, 189710,11,35,36. The two latter species are endemic in the Yucatan Peninsula. O. phyllotis and P. yucatanicus have been adapted to laboratory and a colony for experimental studies has been established at our institution, however the former has been rejected to be employed as an experimental model to study CL caused by L. (L.) mexicana14. The use of local mammalian fauna as a model host to study the development of experimental Leishmania spp. infection could give a more approximate whole picture of what probably occurs in natural conditions12.

In order to develop a novel experimental model of LCL caused by L. (L.) mexicana we sought to characterize clinical and histological features of P. yucatanicus experimentally infected with L. (L.) mexicana as a fundamental first step for developing an infection model.

MATERIALS AND METHODS

Animals: Six month old P. yucatanicus of both sexes were selected from the colony. The animals were maintained in the animal care facility of the Centro de Investigaciones Regionales, Universidad Autonóma de Yucataán (CIR, UADY). The mice were housed individually in small cages (19 x 29 x 12 cm) lined with wood shavings and fed ad libitum rodent chow (2018S Harlan, Madison, Wisconsin, USA). For enrichment and food balance, once a week the mice were given fresh fruit or vegetables. During the experiment, the animals were kept at 22 °C with a 12/12h light cycle. Physical enrichment was provided weekly in the form of cardboard tubes for hiding and paper for nesting material. All animals were euthanized with sodium pentobarbital 100 mg/kg, according to the Mexican Law for the use of laboratory animals, and the Guide for the Care and Use of Laboratory Animals13,29. The proposal was approved by the bioethics committee of CIR, UADY.

Parasite: Although the dose of Leishmania promastigotes delivered by the sand fly vector to the skin of mammalian host has never been directly determined, using a real time-PCR approach it has been possible to reveal a remarkable range of 10 to 100,000 in the dose of parasites transmitted to mice exposed to a single infected sand fly15. More recently, another study determined infection parameters in the sand fly vector that predict transmission of L. (L.) major: it was estimated in the range of 103 to 106 total parasites per fly34. Taking into account these studies we chose to inoculate 1x106 promastigotes to increase the possibility of inducing CL clinical signs.

Local strain MPERO/MX/97/Py4, isolated from a P. yucatanicus with clinical signs (ulcerated lesion), was identified by isoenzymes and monoclonal antibodies as L. (L.) mexicana and was selected for use in this study9,10. Its infectivity was restored by a passage in Syrian golden hamsters (Mesocricetus auratus). Promastigotes were grown in Senekjie's medium for seven days at 23 ºC. Stationary-phase promastigotes were washed three times in RPMI-1640 medium (Gibco) before being counted and adjusted to a 1x106 concentration for inoculation at a final volume of 40 µL. All animals were inoculated in the base of the tail.

Parasite demonstration: Based on previous studies in which local signs of infection by L. (L.) mexicana were always located at the base of the tail10,11,35,36, smears were made from samples collected at the edge of the lesion (avoiding ulcerated and/or heavily crusted parts) or from the inoculation site in absence of local signs. Moreover, smears were made from the liver and spleen samples. Furthermore, aspirates and cultures from the skin of the base of the tail in each animal were prepared. Liquid culture medium (0.1-0.2 mL RPMI 1640) with antibiotics were inoculated and aspirated repeatedly with an insulin needle (1 mL, 27G x 13mm) subcutaneously into base of the tail or at the margin of the lesion when it existed. The aspirates were cultured into tubes containing modified Senekjie's medium, incubated at 23.5 ºC and examined weekly by light microscope for parasite growth. A culture was considerate positive when at least one promastigote was observed and negative if no parasites were found. If negative, the procedure was repeated until three consecutive aspirates were found with one week intervals.

Experimental infection: A total of 54 P. yucatanicus in groups of 18 animals were inoculated in the base of the tail and were euthanized at three (group I) and six (group II) months post experimental inoculation. Control group (III) was inoculated with RPMI-1640 and euthanized at three months.

Clinical features: All animals were examined weekly for the following local clinical signs of infection including ulcerated lesion, crust and scar, which are the main documented symptoms in humans with LCL caused by L. (L.) mexicana4.

Histological features: Skin biopsies were taken from the edge of the lesion (avoiding ulcerated and/or heavily crusted parts) or from the inoculation site in absence of local signs with a 4-mm disposable punch and 2% xylocaine as an anesthetic just before euthanizing the animals. The material was fixed in neutral formalin, routinely processed and embedded in paraffin. Sections were stained with hematoxylin and eosin. Histopathological findings were grouped in patterns according to a classification proposed by MAGALHAES et al.21. The following five histopathological patterns were defined as having occurred and it has been done in CL patients infected with L. (L.) mexicana: type I) exudative-cellular reaction due to infiltration of histiocytes, lymphocytes, and plasma cells, without granuloma; type II) exudative-necrotic reaction, characterized by cellular infiltration, and necrosis and no granulomatous response; type III) exudative and necrotic-granulomatous reaction corresponding to the pattern described as chronic granulomatous inflammation with or without necrosis; type IV) exudative granulomatous reaction (unorganized granuloma) without necrosis characterized by the presence of an unorganized granulomatous reaction; type V) exudative tuberculoid reaction in which a typical tuberculoid granuloma (organized) is formed.

RESULTS

Clinical features: Local clinical signs were recorded for each one of the experimental groups inoculated with 1x106 promastigotes of L. (L.) mexicana. None of the P. yucatanicus inoculated with RMPI-1640 developed clinical or histological features of Leishmania infection. Absence of local clinical signs was observed in a total of 29/36 (80.55%) of the P. yucatanicus experimentally inoculated including both groups I and II. At three months, Group I, the most common clinical sign was a single ulcerated lesion observed in 27.77% (5/18) P. yucatanicus that completed the study, within a range of eight to 10 weeks post experimental infection and persisting without healing during the whole period of survey. In P. yucatanicus followed for six months, Group II, only in 2/18 (11.11%) ulcerated lesions and crusts were observed, and healing was recorded at week 17 and 18 respectively. Results regarding clinical features are shown in Table 1 and Figure 1. There was no difference in the course of the infection linked to P. yucatanicus gender.

Histological features: At three months the histological picture of Leishmania infection was characterized by pattern type III, i.e. exudative and necrotic-granulomatous reactions corresponding to the pattern described as chronic granulomatous inflammation with or without necrosis were found in 7/7 (100%) biopsies of euthanized P. yucatanicus at three (n = 5) and six (n = 2) months respectively. Parasites were observed in all P. yucatanicus with local signs of infection.

Parasite demonstration: Smears from ulcerated lesions were all positive at three months post experimental inoculation and negative at six months (n = 6 and 2 respectively). Smears from the liver were positive in 11/18 (61.11%) at three months, and in 16/18 (88.88%) at six months. Smears from the spleen were 17/18 (94.44%) positive at three months and 12/18 (66.66%) positive at six months. Isolations and cultures from P. yucatanicus were positive in 61.11% (11/18) and 66.66% (12/18) at three and six months respectively.

DISCUSSION

This is the first study that examines P. yucatanicus clinical and histological features of experimental infection with L. (L.) mexicana. As a preliminary study towards the development of a novel experimental model to study LCL caused by L. (L.) mexicana, a first and fundamental criterion was to document that both clinical and histological features resembled those described previously in humans with LCL caused by L. (L.) mexicana17.

In the present study the most common clinical sign, as reported in patients with LCL4, was a single, ulcerated, rounded small lesion, with elevated borders and necrotic center, observed in 7/36 (19.44%) of the P. yucatanicus experimentally infected with 1x106L. (L.) mexicana promastigotes. Moreover, scars developed in P. yucatanicus gathered the criteria for healed lesions of humans with CL, i.e. round or oval shape, smooth surface, depressed, and hypopigmentation37.

It must be remarked that absence of local clinical signs was observed in a total of 29/36 (80.55%) P. yucatanicus experimentally infected with 1x106L. (L.) mexicana promastigotes. As described previously a criterion to incriminate a primary reservoir of Leishmania stated that the infection in a reservoir species should be relatively nonpathogenic, that is, the infection should not lower the survival possibility of the animal since it has to live until the next transmission season, which is more than seven months in the Peninsula. In other words, the immune system of the reservoir must react to the presence of the parasite in such a manner that while preventing irrevocable damage, the parasite evades sterilizing immunity36.

There was no difference in the course of the infection linked to P. yucatanicus gender. However we do not consider this finding to be conclusive regarding the possible role of sex as demonstrated in the mouse model33. More studies will be done regarding the role of sex in susceptibility and/or resistance to the infection by L. (L.) mexicana in P. yucatanicus.

The histological picture observed in P. yucatanicus experimentally infected with L. (L.) mexicana was predominantly type III with reference to Magalhaes' classification whereas in patients with CL caused by the same Leishmania species it was type III and IV. Although a possible correlation between clinical and histological features has been examined to understand the immunological response in humans infected with L. (L.) mexicana5, it has been difficult to establish because such examination depends on a wide range of variables such as the host, the Leishmania species, the time of evolution at the moment of performing the histological study, the inoculation route and the dose of the parasite.

Parasite isolation and culture from the skin of the base of the tail of P. yucatanicus was positive in 61.11% (11/18) and 66.66% (12/18) at three and six months, and in smears from the liver and the spleen. With reference to this finding L. major DNA was detected by PCR in the lymph nodes, spleens, bone marrow, and livers of C57BL/6 one year after recovery from infection1. Moreover DNA specific for L. (V) braziliensis was detected by PCR in scars after clinical cure of CL24. We have reported in a previous study the retention of L. (L.) mexicana in 29 naturally infected rodents including six P. yucatanicus. These cricetids lived in captivity for up to two years and were tested monthly for the presence of the parasite by cultures of needle aspirates from the base of the tail35. These data support the persistence of the parasite in P. yucatanicus experimentally infected, as in the human treated or spontaneously healed and in the mouse model of CL.

Since the 1970s parasitic protozoa of the genus Leishmania have provided a useful perspective for immunologists in terms of host defense mechanisms critical for the resolution of infection caused by intracellular pathogens in experimental studies done in the mouse model. The paradigm for the CD4+ T-helper 1 (Th1)/Th2 dichotomy is largely based, respectively, to the Leishmania major infection in a susceptible/resistant mouse model. Different virulence factors have been identified for distinct Leishmania species, and there are profound differences in the immune mechanisms that mediate susceptibility/resistance to infection and in the pathology associated with disease. These variations not only point to interesting features of the host-pathogen interaction and immunobiology of this genus of parasitic protozoa, but also have important implications for immunotherapy and vaccine development22.

A view of leishmaniasis that only considers mouse model infection with L. major misses a wealth of interesting immunobiology associated with other species of Leishmania. L. (L.) amazonensis and L. (L.) mexicana are associated with quite different patterns of disease following infection of mice when compared with L. major. For example, C57BL/6 or C3H mice, which heal from the L. major infection, develop chronic disease when infected with either L. (L.) amazonensis or L. (L.) mexicana2. Conflicting data may arise in part because different parasite strains or species are being examined, different tissue target (the mice's footpad, the ear, or base of the tail) are being infected, and different doses ("low" 1x102 and "high" 1x106) of metacyclic promastigotes have been inoculated20,27,28. In addition immunological studies carried out in humans did not reproduce the Th1/Th2 dichotomy described in the L. major, C57BL/6 and the BALB/c mouse model of CL18,23,24. In summary there is not an experimental model to study LCL caused by L. (L.) mexicana.

Although classical laboratory mice have advantages, such as colony maintenance, breed all year round, and a short generation time of 10-12 weeks, they have serious limitations when compared with human populations, e.g. they do not exhibit a great variety of natural genetic polymorphisms12.

A new approach to study host-parasite relationships bring out because no animal model was available to study the Sin Nombre virus (SNV), a Hantavirus which is a rodent-borne pathogen with a worldwide distribution and that can cause hantavirus cardiopulmonary syndrome (HCPS) in North America. This situation led to the experimental infection of the deer mouse Peromyscus maniculatus, a primary reservoir of the SNV8. However as in the golden hamster experimental model to study visceral leishmaniasis research is hindered by the lack of commercially available immunological and molecular reagents to investigate mechanisms of disease25.

In summary, we have found that both clinical and histological features of P. yucatanicus infected experimentally with L. (L.) mexicana resembled our previous studies done in patients with LCL caused by this parasite. Moreover parasite persistence was recorded as it has been described in patients where CL healed spontaneously after treatment1. These results support the utility of P. yucatanicus as a novel experimental model to study LCL caused by L. (L.) mexicana.

ACKNOWLEDGMENTS

We thank the personnel from the animal-house of the Centro de Investigaciones Regionales "Dr. Hideyo Noguchi", Universidad Autonoma de Yucatán; Dr. Tony Schountz, for editing the manuscript; IQ Silvia Andrade Canto for photography technical assistance.

13. Guide for the care and use of laboratory animals. Institute of Laboratory Animal Resources Commission on life Sciences. National Research Council. Washington: National Academy Press; 1996. Available from: http://www.nap.edu/openbook.php?record_id=5140. [ Links ]

22. McMahon-Pratt D, Alexander J. Does the Leishmania major paradigm of pathogenesis and protection hold for New World cutaneos leishmaniases or the visceral disease? Immunol Rev. 2004;201:206-24. [ Links ]

27. Menon JN, Bretscher PA. Characterization of the immunological memory state generated in mice susceptible to Leishmania major following exposure to low doses of L. major and resulting in resistence to a normally pathogenic challenge. Eur J Immunol. 1996;26:243-9. [ Links ]